1. Field of the Invention
The present invention relates generally to the field of molecular biology and biochemistry. More particularly, it concerns methods and compositions for expression of inhibitory RNA molecules.
2. Description of Related Art
RNA interference (RNAi) is a naturally occurring, evolutionary conserved biological process in plants and animals as well as humans. Suppression of gene expression via RNAi occurs either by sequence specific degradation of mRNA molecules, or by interfering with translation of mRNA molecules. These two mechanisms are mediated by small interfering RNA (siRNA) molecules that recognize their complimentary sequences in the mRNA. To describe the natural RNAi pathway in brief, an RNAi gene when transcribed produces a primary micro RNA molecule (pri-miRNA). In the nucleus, the enzyme Drosha recognizes structural elements of the pri-miRNA and cleaves it accordingly to produce a premature mi-RNA (pre-miRNA). Exportin 5 transports the pre-miRNA into the cytoplasm where the enzyme Dicer further cleaves the molecule resulting in a miRNA duplex. One half i.e. one strand of the miRNA duplex is then selected and incorporated into the RNA-induced silencing complex (RISC). In most cases, if the strand of the miRNA duplex selected is 100% complimentary to the target mRNA, cleavage of the mRNA occurs. If the strand of the miRNA duplex selected is not 100% complimentary to the target mRNA, mRNA may not be degraded but RISC will interfere with ribosome function, hence decreasing translation of the protein. In both cases, the resulting decrease of final protein synthesis is termed post-transcriptional gene silencing (PTGS) (He and Hannon, 2004).
Down-regulation of gene expression by use of RNAi is now also being explored as a promising therapeutic tool to address infectious diseases, cancers, as well as inheritable genetic diseases. However, despite significant advances in the field, the magnitude of reduction in gene expression that is achieved with such strategies is often insufficient to provide clinical efficacy. Methods for significant and sustainable targeted reduction of gene expression are therefore still in great need.